Abnormal cellular proliferation, notably hyperproliferation, is the source of numerous diseases, the most severe one being cancer. In the United States alone approximately 1.5 million people are diagnosed with cancer and 0.5 million die from it each year. The fight against cancer has seen some success but also numerous set-backs. There is a great need for innovative therapeutics.
Targeted drug delivery, as opposed to systemic delivery, can dramatically increase drug efficacy while decreasing side effects. Targeted delivery requires a target (antigen, receptor), a delivery vehicle (cytokines, antibodies and fragments thereof) and a drug. Currently, several strategies are used for delivering drugs to targets: they are based either on direct conjugates to the targeting protein or on derivatized carriers that interact with specific adapters that are conjugated to the targeting protein. Recently, heterobifunctional recombinant antibodies recognizing one epitope on the cell surface and another epitope on the drug carrier have been proposed for targeted drug delivery. However, these constructs only make contact with one binding site and therefore show high dissociation rates. The strong interaction of streptavidin with biotin has been particularly widely explored for a targeting approach. Unfortunately, streptavidin is highly immunogenic and furthermore shows an inherent high kidney accumulation.
Over the last few years, a burst of reports on the construction of multivalent recombinant antibody fragments has appeared (Pluckthun, A. and Pack, P. Immunotechnology 3, 83-105, 1997. Todovroska, A. et all., J. Immunol. Methods 248, 47-66, 2001). Multivalency not only enhances the strength of binding, but it also amplifies binding selectivity. These designs have included a variety of recombinant fusions using adhesive protein domains, peptides or specially designed linkers for formation of single chain antibody fragment multimers. For medical applications such as targeting to tumor-associated antigens, efficient tissue penetration must be combined with high functional affinity, and fragments must be stable against denaturation or proteolysis until they have reached the tumor site. Most of these published constructs will not meet all of these prerequisites: multimeric constructs assembled by domains or peptides show dissociation at dilution, multimeric constructs assembled by specific linkers are non-homogeneous products and the production of all of these constructs is hampered by aggregation and subsequent precipitation of the proteins at high protein concentration.
The invention presents a novel method for the design of multimers based on the ribonuclease barnase and its inhibitor, barstar. The complex between barnase and barstar is extremely tight with a Kd˜10−14 M and forms very rapidly, comparable in affinity with the streptavidin/biotin system (Hnatowich et al., J. Nucl. Med. 28: 1294-1302, 1987).
The barnase and barstar proteins are small (110-residues for barnase and 89-residues for barstar), stable, very soluble and resistant to proteases—features commensurate with bacterial expression. Moreover, the three-dimensional structure of the complex is known both from X-ray crystallography3 and NMR spectroscopy4,5 and shows that the N-terminal as well C-terminal parts of both proteins localize outside of the barnase:barstar interface. They are therefore accessible for fusion with targeting proteins and then form extremely stable multimers due to the practically irreversible pairing of these ligands.
In addition barnase and barstar have been used in genetically engineered plants. It has been shown that male fertility can be restored to the plant with a chimeric fertility-restorer gene comprising another DNA sequence (or fertility-restorer DNA) that codes, for example, for a protein that inhibits the activity of the cytotoxic product or otherwise prevents the cytotoxic product to be active in the plant cells (European patent publication “EP” 0,412,911). For example the barnase gene of Bacillus amyloliquefaciens codes for an RNase, the barnase, which can be inhibited by a protein, the barstar, that is encoded by the barstar gene of B. amyloliquefaciens. The barnase gene can be used for the construction of a sterility gene while the barstar gene can be used for the construction of a fertility-restorer gene. Experiments in different plant species, e.g. oilseed rape, have shown that a chimeric barstar gene can fully restore the male fertility of male sterile lines in which the male sterility was due to the presence of a chimeric barnase gene (EP 0,412,911, Mariani et al., Proceedings of the CCIRC Rapeseed Congress, Jul. 9-11, 1991, Saskatoon, Saskatchewan, Canada; Mariani et al., Nature 357: 384-387, 1992,). By coupling a marker gene, such as a dominant herbicide resistance gene (for example the bar gene coding for phosphinothricin acetyl transferase (PAT) that converts the herbicidal phosphinothricin to a non-toxic compound (De Block et al., EMBO J. 6:2513, 1987), to the chimeric male-sterility and/or fertility-restorer gene, breeding systems can be implemented to select for uniform populations of male sterile plants (EP 0,344,029; EP 0,412,911).
Barnase and barstar have been used in a new approach for effective positive selection during gene manipulation. Several plasmid vectors for molecular cloning were constructed. They are based on the expression plasmid for a bacterial ribonuclease, barnase. In addition to the barnase gene under control of a synthetic tac promoter, these plasmids carry the gene for the barnase inhibitor, barstar, the constitutive expression of which protects the bacterium from the detrimental effects of moderate barnase production. Full expression of the barnase gene overcomes protection by barstar and becomes lethal. The entire pUC polylinker was inserted into the barnase gene for convenient cloning of genes of interest. Uncut or religated vectors will preclude growth while plasmids with inserts in the barnase gene will let the cells survive. The resulting plasmids are generally useful selective cloning vectors representing the <<kill-the-rest>> approach for studies in molecular biology and biotechnology (RU 2105064 C1, 1996).